Partial silencing of a hydroxy-methylglutaryl-CoA reductase-encoding gene in Trichoderma harzianum CECT 2413 results in a lower level of resistance to lovastatin and lower antifungal activity

In the present article, we describe the cloning and characterization of the Trichoderma harzianum hmgR gene encoding a hydroxymethylglutaryl CoA reductase (HMGR), a key enzyme in the biosynthesis of terpene compounds. In T. harzianum, partial silencing of the hmgR gene gave rise to transformants with a higher level of sensitivity to lovastatin, a competitive inhibitor of the HMGR enzyme. In addition, these hmgR-silenced transformants produced lower levels of ergosterol than the wild-type strain in a minimal medium containing lovastatin. The silenced transformants showed a decrease in hmgR gene expression (up to a 8.4-fold, after 72h of incubation), together with an increase in the expression of erg7 (up to a 15.8-fold, after 72h of incubation), a gene involved in the biosynthesis of triterpenes. Finally, hmgR-silenced transformants showed a reduction in their antifungal activity against the plant-pathogen fungi Rhizoctonia solani and Fusarium oxysporum.     

Immunological identity of a 60 kd oncofetal protein induced in rats by chemical carcinogens and released by transformed cells

A 60,000 dalton (60 kd) oncofetal protein was previously shown to be produced by tumors in tumor-bearing rats and by target tissues within 3 weeks of carcinogen treatment. The factor is released to and accumulates in the blood in vivo and in the conditioned medium of cultured transformed cells in vitro. A polyclonal antibody produced against the 60 kd factor purified from the plasma of a rat carrying the N-2-fluorenylphthalamic acid-induced transplantable Hepatoma 7777, was tested against the 60 kd factor from various sources. Based on the results of immunoprecipitation of biochemical activity associated with the 60 kd factor, it was determined that these anti-60 kd antibodies cross-reacted with the factor released by a dimethylbenzanthracene-induced rat mammary carcinoma, with the factor in rat tumor cytosol and with rat spontaneous lymphoma cells, but not with a 60 kd factor isolated from pooled cancer patient plasma. Furthermore, these antibodies cross-reacted with the 60 kd factor induced within 21 days of treatment of the rats with a range of carcinogens from 8 chemical structural groups. The anti-60 kd factor antibodies did not cross-react with a 35 kd factor having similar biochemical activity found in normal adult cells.     

HLF/HIF-2alpha is a key factor in retinopathy of prematurity in association with erythropoietin

An HLF (HIF-1alpha-like factor)/HIF-2alpha-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF(kd/kd)) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF(kd/kd) mice expressing 80-20% reduction, depending on the tissue, in wild-type HLF mRNA were fertile and apparently normal. Hyperoxia-normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF(kd/kd) mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF(kd/kd) mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF(kd/kd) mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF(kd/kd) mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.     

lack of suppressor cell activity for natural killer cells in infant, aged and a low responder strain of mice

We have examined the reported role of suppressor cells in the regulation of NK activity in mice with naturally low NK activity (infant and aged (C57 X A)F1 hybrids (CAF1) and low responder strain AKR mice). Possible suppressor activity was assayed by mixing, at a 1 : 1 ratio, spleen cells from low activity mice with spleen effector cells from normally active 8 to 10 wk old CAF1 mice. The lytic activity of the mixture was compared with the activity of effector cells diluted with medium alone or diluted 1 : 1 with "non-suppressor" population which served as a control for nonspecific decreases in lysis. The control or "filler" cells employed were suspensions of adult CAF1 thymus, thymus from adult mice exposed to 2,000 R, and adult CAF1 spleen cells cultured for 24 hours, a procedure that depleted NK activity. In no case was the activity observed in the presumed suppressor-effector mixture significantly lower than that observed in the filler-effector cell mixtures. Thus, in infant (1 to 2 wk) and aged (12 to 18 mo) CAF1 mice and in 8 to 10 wk old AKR mice, we found no evidence for specific cell-mediated suppression of natural cytotoxicity.     

Maspin is an angiogenesis inhibitor

Maspin, a unique member of the serpin family, is a secreted protein encoded by a class II tumor suppressor gene whose downregulation is associated with the development of breast and prostate cancers. Overexpression of maspin in breast tumor cells limits their growth and metastases in vivo. In this report we demonstrate that maspin is an effective inhibitor of angiogenesis. In vitro, it acted directly on cultured endothelial cells to stop their migration towards basic fibroblast growth factor and vascular endothelial growth factor and to limit mitogenesis and tube formation. In vivo, it blocked neovascularization in the rat cornea pocket model. Maspin derivatives mutated in the serpin reactive site lost their ability to inhibit the migration of fibroblasts, keratinocytes, and breast cancer cells but were still able to block angiogenesis in vitro and in vivo. When maspin was delivered locally to human prostate tumor cells in a xenograft mouse model, it blocked tumor growth and dramatically reduced the density of tumor-associated microvessels. These data suggest that the tumor suppressor activity of maspin may depend in large part on its ability to inhibit angiogenesis and raise the possibility that maspin and similar serpins may be excellent leads for the development of drugs that modulate angiogenesis.     

Characterization of a suppressor mutation complementing an acid-sensitive mutation in Streptococcus mutans

We isolated a spontaneous suppressor mutant complementing the acid-sensitive phenotype of Streptococcus mutans strain Tn-1, a mutant previously generated in this laboratory, defective in the activity of the dgk-encoded putative undecaprenol kinase. A relatively simple genetic method was developed to identify the suppressor mutation, based on selection for transformants containing two closely linked markers: a selectable allele of the unknown suppressor gene and an antibiotic resistance gene introduced on a suicide plasmid at random sites into the chromosome via homologous recombination. While we have not actually identified the original suppressor mutation, another mutated gene restoring acid resistance has been isolated, which suggests a possible mechanism of suppression.     

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.     

Deletion of the yhhP gene results in filamentous cell morphology in Escherichia coli

The Escherichia coli yhhP gene was predicted to encode a small hypothetical protein of 81 amino acids, the cellular function of which is not known. To gain insight into the function of this uncharacterized YhhP protein, genetic and biochemical studies were done. We first tried to express and purify the YhhP protein to prepare an anti-YhhP antiserum. Western blotting showed that the hypothetical yhhP gene is indeed transcribed and translated as a minor cytoplasmic protein. YhhP-deficient (delta yhhP) cells formed colonies poorly on a rich medium (e.g., Luria-Bertani medium) containing a relatively low concentration of NaCl, while they can grow normally either in LB containing 3% NaCl or in a synthetic medium (e.g., M9-glucose). During exponential growth in rich medium, an early step of cell division was inhibited in delta yhhP cells, forming filaments. For the YhhP-deficient filamentous cells, the FtsZ-ring formation was analyzed with immunofluorescence microscopy. The FtsZ-ring formation did not occur normally in the delta yhhP filaments, although the filamentous cells contained the FtsZ protein at a certain level comparable to that in the wild-type cells. The ftsZ gene was found to function as a multicopy suppressor of the delta yhhP mutant. Another multicopy suppressor gene was identified as the dksA gene. Provided that either the ftsZ or dksA gene was introduced into the mutant cells with its multicopy state, the resulting transformants were capable of growing in rich medium, formed wild-type short rods. These results are discussed with regard to the presumed function of this ubiquitous protein.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis

In Xenopus, a cytoplasmic agent known as MPF induces entry into mitosis. In fission yeast, genetic studies have shown that the cdc2 kinase regulates mitotic initiation. The 13 kd product of the suc1 gene interacts with the cdc2 kinase in yeast cells. We show that the yeast suc1 gene product (p13) is a potent inhibitor of MPF in cell-free extracts from Xenopus eggs. p13 appears to exert its antagonistic effect by binding directly to MPF. MPF activity is quantitatively depleted by chromatography on a p13 affinity column. Concomitantly, the Xenopus counterpart of the yeast cdc2 protein is adsorbed to the column. A 42 kd protein also binds specifically to the p13 affinity matrix. These findings suggest that the Xenopus cdc2 protein and the 42 kd protein are components of MPF.     

Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.     

Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity.

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.     

Cytotype control of Drosophila P element transposition: the 66 kd protein is a repressor of transposase activity

Drosophila P transposable elements encode two proteins, an 87 kd transposase protein and a 66 kd protein that has been hypothesized to repress transposition. We have made germline transformants carrying modified P element derivatives that encode only the 66 kd protein and shown that these elements repress transposase activity in both the germline and the soma. The position of these elements in the genome quantitatively affected their ability to negatively regulate transposase and to express the 66 kd protein. Single 66 kd element-containing strains did not exhibit the maternal inheritance of P cytotype characteristic of P strains. However, we demonstrated that a true P strain produced higher levels of the 66 kd protein during oogenesis than single 66 kd P elements. Thus, the expression of the 66 kd repressor during oogenesis may be a major determinant of the maternal effect of P cytotype.     

苏云金芽孢杆菌以色列亚种130kd杀蚊蛋白基因的...

The location of 130kd mosquitocidal protein gene of Bti 4Q5 strain on its 75Md plasmid was confirmed by southern hybridization using a 18-base oligonucleotide probe. The crystal protein containing the component of 130kd toxic protein was purified. The crystal protein exhibiting the mosquitocidal activity against larvae of Aedes aegypti was shown by bioassay. The purified 75Md plasmid DNA of Bti 4Q5 strain was completely digested with HindIII restriction enzyme, ligated with the vector pUC18 and transformed into the recipient cells of E. coli TG1. From Apr transformants, four clones with HindIII restriction fragment inserts highly homologous to the 18-base oligonucleotide probe were obtained by in situ hybridization and southern hybridization. The 5.2kb HindIII restriction fragment insert was obtained in clone pFH2 and clone pFH4, and 2.3kb HindIII restriction fragment insert in clone pFH1 and pFH3. For pFH2 and pFH4, the 5.2kb fragment was inserted in pUC18 in opposite orientation. It contained 130kd mosquitocidal protein gene (type I) identified by restriction enzyme map analysis. The 2.3kb HindIII fragment insert in other two clones (pFH1 and pFH3) harbored a part of the type II mosquitocidal protein gene which can be used as a probe for cloning of the type II mosquitocidal protein gene.     

Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue

An antiserum against material shed into serum-free medium by MCF-7 human mammary carcinoma cells (anti-SFM II) disrupts cell-cell interactions in murine mammary tumor epithelial cells (MMTE). We now report purification of an 80 kd glycoprotein (GP80) from SFM of MCF-7 mammary carcinoma cells that blocks the activity of anti-SFM II. Anti-SFM II also inhibits compaction of eight-cell mouse embryos, and purified GP80 blocks this reaction. An antiserum against purified GP80 (anti-GP80) has all adhesion-disrupting activities displayed by anti-SFM II. It recognizes one band at 80 kd in SFM and a 120 kd band in detergent extracts of epithelial but not fibroblastic cells. In immunofluorescence studies it is restricted to sites of cell-cell interaction in cultured epithelial cells. Thus a cell surface glycoprotein of 120 kd, the medium form of which is approximately 80 kd, which is neither species nor tissue specific, is expressed at early stages of mammalian development and is found on epithelia.     

BRG1 is a prognostic indicator and a potential therapeutic target for prostate cancer

Brahma-related gene 1 (BRG1) is one of two mutually exclusive ATPases that function as the catalytic subunit of human SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling enzymes. BRG1 has been identified as a tumor suppressor in some cancer types but has been shown to be expressed at elevated levels, relative to normal tissue, in other cancers. Using TCGA (The Cancer Genome Atlas) prostate cancer database, we determined that BRG1 mRNA and protein expression is elevated in prostate tumors relative to normal prostate tissue. Only 3 of 491 (0.6%) sequenced tumors showed amplification of the locus or mutation in the protein coding sequence, arguing against the idea that elevated expression due to amplification or expression of a mutant BRG1 protein is associated with prostate cancer. Kaplan-Meier survival curves showed that BRG1 expression in prostate tumors inversely correlated with survival. However, BRG1 expression did not correlate with Gleason score/International Society of Urological Pathology (ISUP) Grade Group, indicating it is an independent predictor of tumor progression/patient outcome. To experimentally assess BRG1 as a possible therapeutic target, we treated prostate cancer cells with a biologic inhibitor called ADAADi (active DNA-dependent ATPase A Domain inhibitor) that targets the activity of the SNF2 family of ATPases in biochemical assays but showed specificity for BRG1 in prior tissue culture experiments. The inhibitor decreased prostate cancer cell proliferation and induced apoptosis. When directly injected into xenografts established by injection of prostate cancer cells in mouse flanks, the inhibitor decreased tumor growth and increased survival. These results indicate the efficacy of pursuing BRG1 as both an indicator of patient outcome and as a therapeutic target.     

Synthesis of isoflavene-thiosemicarbazone hybrids and evaluation of their anti-tumor activity

Phenoxodiol is an isoflavene with potent anti-tumor activity. In this study, a series of novel mono- and di-substituted phenoxodiol-thiosemicarbazone hybrids were synthesized via the condensation reaction between phenoxodiol with thiosemicarbazides. The in vitro anti-proliferative activities of the hybrids were evaluated against the neuroblastoma SKN-BE(2)C, the triple negative breast cancer MDA-MB-231, and the glioblastoma U87 cancer cell lines. The mono-substituted hybrids exhibited potent anti-proliferative activity against all three cancer cell lines, while the di-substituted hybrids were less active. Selected mono-substituted hybrids were further investigated for their cytotoxicity against normal MRC-5 human lung fibroblast cells, which identified two hybrids with superior selectivity for cancer cells over normal cells as compared to phenoxodiol. This suggests that mono-substituted phenoxodiol-thiosemicarbazone hybrids have promising potential for further development as anti-cancer agents.